Capillary and microchip electrophoresis for the analysis of small biomolecules

CAPILLARY AND MICROCHIP ELECTROPHORESIS FOR THE ANALYSIS OF SMALL BIOMOLECULES ELAINE TAY TENG TENG NATIONAL UNIVERSITY OF SINGAPORE 2008... SUMMARY Capillary electrophoresis and its m

CAPILLARY AND MICROCHIP ELECTROPHORESIS FOR THE ANALYSIS OF SMALL BIOMOLECULES

ELAINE TAY TENG TENG

NATIONAL UNIVERSITY OF SINGAPORE

2008

CAPILLARY AND MICROCHIP ELECTROPHORESIS FOR

THE ANALYSIS OF SMALL BIOMOLECULES

ELAINE TAY TENG TENG (B.Sc (Hons), NUS)

A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE

2008

ACKNOWLEDGEMENTS

Foremost, I would like to express my gratitude to my supervisor, Prof Sam Li Fong Yau For the past few years, he had showered me with encouragement and valuable advices in various aspect of my MSc work despite his hectic schedule In addition, Prof Li had provided me with plenty of opportunities to acquire new analytical and instrumental skills as well as encouraged me to attend overseas conferences to gain greater exposures to the research arena For all these, I am grateful for his support

My enriching and pleasant MSc research experience was also attributed to the guidance and assistance provided by my mentors and lab mates in Prof Sam Li’s research group I would like to express special appreciation to Wai Siang, Hiu Fung and Guihua who had often set aside time to discuss and troubleshoot tricky problems encountered during my MSc research work Their jokes and laughter provided me relief during this stressful period

I would also like to express my immense gratitude to my friends, Seah Ling and Kim Huey, and my family for their love, understanding and moral support throughout the course of my studies They listened to my grumbles patiently and had been tolerant with my long working hours in research lab

Last but not least, I would also like to show my appreciation to NUS for providing me with a research scholarship that had financed my study throughout my MSc research term My heartfelt thanks to the NUS technical staff from CMMAC, Lab Supply and Department of Chemistry, in particularly Mdm Frances, Ms Tang, Mdm Han, Suria and Agnes, for aiding me in various aspects of my MSc research project and administrative work

Table of Contents Acknowledgements I Table of Contents II Summary VI List of Tables VIII List of Figures IX List of Schemes XI List of Symbols XII

CHAPTER 1 Electrophoresis of Small Biomolecules 1

1.1 Principles of capillary electrophoresis 1

1.2 Microchip capillary electrophoresis 5

1.3 Analysis of small biomolecules 10

1.4 Project Objectives 12

1.5 References 13

CHAPTER 2 Analysis of Adenosine 16

2.1 Importance of the adenosine analysis 16

2.2 Liquid-liquid extraction of adenosine 17

2.2.2 Target specific liquid-liquid extraction with ionic liquid-aptamer 20

2.3 Experimental 23

2.3.1 Materials and apparatus 23

2.3.1.1 Instrumentation 23

2.3.1.2 Reagents and chemicals 23

2.3.2 Microwave Synthesis of 1-butyl-3-methylimidazolium chloride 24

2.3.3 Analysis of 1-butyl-3-methylimidazolium based ionic liquid via CE-UV 25

2.3.4 Synthesis of 1-butyl-3-methylimidazolium based ionic liquid 25

2.3.5 Synthesis of 1-butyl-3-methylimidazolium hexafluorophosphate 27

2.3.6 Liquid-liquid extraction of adenosine using 1-butyl-3-methylimidazolium based ionic liquid-42-mer extractant 27

2.3.7 CE-UV analysis of adenosine 28

2.4 Results and Discussion 29

2.4.1 DNA aptamer of adenosine 29

2.4.2 Synthesis of 1-butyl-3-methylimidazolium-2’-deoxycytidine-5’-

monophosphate 30

2.4.3 Liquid-liquid extraction of adenosine using 1-butyl-3-methylimidazolium based ionic liquid-42-mer extractant   34

2.5 Conclusion   41

2.6 References   43 

CHAPTER 3 Floating Resistivity Detector for Microchip

Electrophoresis 45

3.1 Microchip and its detection modes 45

3.2 Conductimetry – universal detection method 49

3.3 Floating resistivity detector (FRD) 52

3.4 Working principles of floating resistivity detector 53

3.5 Experimental 56

3.5.1 Materials and apparatus 56

3.5.1.1 Instrumentation 56

3.5.1.2 Reagents and chemicals 56

3.5.2 Fabrication of microchip 57

3.5.3 Designing and optimization of FRD microchip 58

3.5.4 Standard microchip electrophoresis procedures 60

3.6 Result and Discussion 61

3.6.1 Optimized microchannel layout of FRD microchip 61

3.6.2 Applications of FRD 64

3.6.2.1 Metal cations analysis 64

3.6.2.2 Amino acids analysis 66

3.6.2.3 Biogenic amines analysis 67

3.7 Conclusion 69

3.8 References 70

CHAPTER 4 Concluding Remarks 73

Appendices 76

SUMMARY

Capillary electrophoresis and its miniaturized counterpart, microchip capillary electrophoresis are becoming increasingly popular analytical techniques among the research groups due to the simple instrumental set-up, high throughput sensitive analysis as well as low reagents and sample consumption while allowing analysis of various analytes to reach up to ultra-trace level

Thus, such analytical techniques are apt for the analysis of small biomolecules The quantitative analysis of small biomolecules in the body system allows better understanding of a patient’s health since any health deterioration can be accompanied

by an abnormal changes in the level of these small biomolecules However, these small biomolecules are present in small amount in the human body such that their analyses are often laborious due to the need for extensive sample preparation and sensitive detection method Such an analysis also impedes routine analysis However, with the high separation efficiency that can be expected from capillary electrophoresis and its miniaturized counterpart, it can allow more analyses to be carried out on these small biomolecules Hence, a study with capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) was chosen to be carried out on these biomolecules

extraction for adenosine, a biomarker for inflammatory diseases and cell stress Various methods of obtaining the ionic liquid-aptamer based extractant were attempted The structure and quantity obtained were subsequently analyzed via various spectroscopic methods as well as capillary electrophoresis The extraction efficiency of these extractants was then examined with a capillary electrophoresis system coupled to an ultraviolet/visible detector due to adenosine’s UV-absorbing nature

In view of the non-UV absorbing property of many small biomolecules like amino acids and biogenic amines as well as the need for rapid analysis, a novel contact conductivity detection system for microfluidic devices was developed This detector served to provide a universal mode of detection while the microfluidic device aided in enhancing the analytical throughput Its detection principle was similar to most conductivity detectors except that it measured with its “liquid electrode voltage probes” that minimized fouling of the detection electrode surface and thereby increasing the repeatability of analysis Its analytical performance was consequently evaluated with simple metal ions as well as in the separation of amino acids and biogenic amines

LIST OF TABLES

Page

Table 2.1 The extraction efficiency of [C4MIM] based ionic liquid and [C4MIM]

based ionic liquid-42-mer extractants for adenosine in aqueous sample 39

Table 2.2 The extraction efficiency of [C4MIM] based ionic liquid and [C4MIM]

based ionic liquid-42-mer extractants for adenosine and its analogues

in aqueous sample 41

Table 3.1 The limits of detection of various modes of detection in MCE 46

Table 3.2 The parameters and their respective conditions in the stepwise

optimization of the dimensions of the microchip detection window 59

Table 3.3 The resolution between the respective peaks in the stepwise optimization

of the length between detection probe and buffer waste reservoir 62

LIST OF FIGURES

Page

Figure 1.1 Schematic diagram depicting the basic setup of a capillary

electrophoresis system 2

Figure 2.1 Representative cations used in the synthesis of ionic liquids 19

Figure 2.2 Chemical structures of [C4MIM]OH and four nucleotides 22

Figure 2.3 Molecular recognition section of the 42-mer of adenosine 30

Figure 2.4 Chemical structure of adenosine 30

Figure 2.5 Electrophereogram of varying concentrations of methylimidazole and synthesized [C4MIM]OH 33

Figure 2.6 Electrophereogram of adenosine, dimethylsulfoxide and 42-mer 36

Figure 2.7 Electrophereogram of adenosine, blank water and two-fold acetonitrile diluted ionic liquid layer after extraction of adenosine 36

Figure 2.8 Electrophereogram of adenosine and cytosine in various solvents 37

Figure 2.9 Chemical structures of adenosine and its analogues 41

Figure 3.1 Schematic diagram depicting the arrangement of the microelectrodes on the microchannel 50

Figure 3.2 Schematic diagram of the circuit of the floating resistivity detector microchip capillary electrophoresis system 53

Figure 3.3 Schematic diagram of the floating resistivity detector microchip 59

Figure 3.4 The peak intensity and resolution between the respective peaks in the optimization of the length of the detection probe, Parameter 2 63

Figure 3.5 The peak intensity and resolution between the respective peaks in the

optimization of the length of the detection window, Parameter 3 64

Figure 3.6 Electrophereogram of 4 metal cation standards determined by

microchip electrophoresis with floating resistivity detector 65

Figure 3.7 Electrophereogram of 4 amino acids determined by microchip

electrophoresis with floating resisitivity detector 67

Figure 3.8 Electrophereogram depicting the effect of separation voltage on the

separation of biogenic amines 68  

LIST OF SCHEMES

Page

Scheme 2.1 Acid-base reaction between [C4MIM]OH and 42-mer of adenosine 31

Scheme 2.2 Acid-base reaction between [C4MIM]OH and

2’-deoxycytidine-5’-monophosphate 33

BW: Buffer waste reservoir

C 4 D: Capacitively coupled contactless

conductivity detector

CCD: Contact conductivity detector

CE: Capillary electrophoresis

CGE: Capillary gel electrophoresis

CIEF: Capillary isoelectric focusing

COC: Cyclic olefin copolymer

DA: “liquid electrode voltage probe” A

DAQ: Data acquisition

DB: “liquid electrode voltage probe” B

EA: Ethyl acetate ECEEM: Equilibrium capillary electrophoresis equilibrium mixture

EOF: Electroosmotic flow ESMC: Electrolyte solution mediated

ILs: Ionic liquids IPA: Isopropyl alcohol ISE: Ion-selective electrode LC-MS: Liquid chromatography –

mass spectrometry

LIF: Laser induced fluorescence LLE: Liquid-liquid extraction LOD: Limit of detection LPME: Liquid phase micro-extraction MALDI-MS: Matrix assisted laser

desorption/ionization-mass

MCE: Microchip capillary

NMR: Nuclear magnetic resonance

PAHs: Polycyclic aromatic

deoxyribonucleic acid

SW: Sample waste reservoir

T g: Glass transition temperature

Tris:Trishydroxymethylaminomethane UV: Ultraviolet

UV-Vis: Ultraviolet-visible VOCs: Volatile organic compounds

CHAPTER 1 Electrophoresis of Small Biomolecules 1.1 Principles of capillary electrophoresis

Capillary electrophoresis (CE) refers to an analytical technique that separates compounds according to their charge-to-size ratios in an aqueous buffer filled fused silica capillary under the influence of an externally applied electric field

Electrophoresis was first described by Tiselius et al.1 in 1930 for the separation of

proteins and Hjerten et al.2 subsequently introduced the first CE setup in 1967

However, CE only sparked off immense interest in the research arena when its simplicity and high separation efficiency was first demonstrated by Lukacs and Jorgensen3 in the separation of small compounds and biomolecules

The CE instrumental system is relatively inexpensive and uncomplicated to set

up as seen in Figure 1.1 below It consists of a high voltage power supply unit (0 – 30 kV), a detector (optical, electrochemical or mass spectrometric) and a computer equipped with a data acquisition (DAQ) software A fused silica capillary, (with inner bore of 25 – 100 of µm wide) together with electrodes from the power supply unit are placed in the sample buffer reservoir and the buffer waste reservoirs, forming a closed electrical circuit When high voltage is applied to the capillary through platinum electrodes, the charged compounds will be attracted to their oppositely charged electrodes As they migrate past the detector placed near the capillary end, peak signals will be registered and recorded against time by the DAQ software in the form

of an electropherogram

Figure 1.1 A schematic diagram depicting the basic setup of a CE system where (a) Platinum

electrodes, (b) Buffer filled capillary, (c) High voltage (HV) Power supply, (d) Detector, (e) Buffer reservoir or sample reservoir during sample injection (f) Buffer waste reservoir and (g) DAQ displaying an electropherogram

In CE, the resultant mobility of each charged compound is dependent on the combinatory effects of the electroosmotic force (EOF) and their respective inherent electrophoretic mobility in the capillary as shown in Equation 1.1:

μeff = μep + μEOF - (1.1) Where μeff refers to the effective electrophoretic mobility of the analyte,

μep refers to the electrophoretic mobility of the analyte as determined by its charge as well as size and

μEOF refers to the electroosmotic mobility of the buffer

The fused silica capillary consists of silanol (Si-OH) groups lining along its inner surface When a solution of pH above 3 is passed through, these silanol groups will be deprotonated, forming negatively charged silanoate (Si-O-) groups A diffuse double film of positively charged buffer cations is electrostatically attacted to these silanoate groups, leading to the formation of the EOF Within this film, a fixed layer of cations

is tightly held to the silanoate groups followed by a mobile layer where the buffer

CE HV Power Supply

(a)

(c) (e)

(g)

(d) (b)

(f)

CE HV Power Supply

(a)

(c) (e)

(g)

(d) (b)

(f) (a)

(c) (e)

(g)

(d) (b)

(f)

voltage is applied, the mobile layer of buffer cations migrates towards the cathode As

it does, it drags the bulk of the buffer solution along with it and thereby generating the EOF The strength of this EOF is determined by Equation 1.2 below:

μEOF = єζ/4πη - (1.2) Where є refers to the dielectric constant of the buffer,

ζ is the zeta potential and

η is the viscosity of the buffer

The buffer parameters are affected by the composition of the buffer used, its pH as well as the type of organic additives introduced For instance, when the pH of the buffer is increased, the zeta potential is high and a strong EOF is resulted However, when an organic additive such as acetonitrile is added, this will raise the buffer’s viscosity and thereby lowering the strength of the EOF

The EOF, thus, determines the times at which the charged compounds migrate out When a strong EOF is generated in normal CE mode, the cations will reach the detector first, followed by the neutrals The anions will also be swept towards the negatively charged electrodes Conversely, when the EOF is weak, the inherent electrophoretic mobilities of the anions will cause them to be attracted to the anode instead

Although CE is not as routinely used as compared to other separation techniques like high performance liquid chromatography (HPLC) and gas chromatography (GC), it is still an attractive technique that draws the attention of researchers For instance, it can attain relatively higher separation efficiency compared with HPLC and GC as its sample plug is electrically driven through the capillary as a flat plug in which all the molecules travel at the same velocity, resulting

in narrow, sharp peaks In addition, the narrow bore of the capillary aids in reducing

band dispersion across the capillary Conversely, the sample in HPLC is pumped through the packed column, of 1 - 10 mm wide, under the laminar flow profile which leads to diffused sample zone and hence broad peaks Moreover, the analysis in CE is not limited to only charged compounds but neutral ones as well The micellar electrokinetic capillary chromatography (MEKC) mode can be applied to such sample analysis in which charged surfactants, introduced in the buffer system, will form micelles which act as pseudo stationary phase to interact with the neutrally charged compounds and thereby influencing their mobilities through the capillary5 Besides these neutral compounds, CE also allows the analyses of a wide variety of analytes in different matrices, for example, inorganic ions in postblast residues6, environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and herbicides7, 8, food additives and organic contaminants (dyes, preservatives and acrylamide)5, 9, 10, pharmaceuticals11 as well as biomolecules12, 13 This can be readily achieved by the application of different modes of CE like microemulsion electrokinetic chromatography (MEEKC), capillary gel electrophoresis (CGE), non-aqueous capillary electrophoresis (NACE) and capillary isoelectric focusing (CIEF) These CE modes can be carried out simply by adjusting the buffer constituents (aqueous or organic solvents), the type of buffer additives used (surfactants and chiral selectors) as well as the concentration and pH of the electrophoresis buffer In addition, CE is a sensitive analytical technique that can analyze up to ultra-trace amounts of analytes in complex sample matrices It is also environmental friendly due to its low reagent consumption and the simplicity of its instrumental setup that allows for automation and portability

1.2 Microchip capillary electrophoresis

With the rapid development of CE in the 1980s, there is a shift in trend in the 1990s towards miniaturization to further exploit its advantages This is in particular so with the first paper reporting on the CE application on a glass microchip fabricated

via photolithographic method by Manz et al.14 in 1992 Since then, there is an increasing number of publications on the various aspects of microchip capillary electrophoresis (MCE) that range from device technology (microfabrication techniques, surface modification, design of the microchip etc) 15-17; analytical methods (sample preparation, detection, separation modes and methods etc)18, 19 and the application areas (immunoassay, clinical diagnosis, cell handling and analysis)20, 21

Despite its small size, microchip CE is still able to achieve high separation efficiency It provides high separation power of up to 160,000 theoretical plates on a

50 μm wide and 20 μm deep microchannel with only a separation length of 50 mm22 With typically short microchannels of 50 - 100 mm long, 10 - 100 μm wide and less than 50 μm deep, only about 1 – 5 kV is required to drive the electrophoresis on microchip23 Hence, Joule heating and consequently dispersive mass transport can be minimized Furthermore, high throughput can also be realized on this small device with μ-capillary array electrophoresis (μ-CAE) The μ-CAE has progressed from the 48-separation lanes24 to as many as 384-separation lanes on a 20 cm wide substrate25

In addition, minimal sample and reagents are required since the sample and buffer reservoirs hold only 50 - 200 μL of solution Thus, it is suitable for the analyses of samples that are precious and available only in limited amounts like proteins, neuropeptides, biogenic amines and amino acids in body fluids like serum and neurological fluids26 Moreover, with the combination of efficient pumping mechanism of electroosmosis and electrophoresis, the integration of various

laboratory functions (sample preparation, mixing, reactors, preconcentration and analysis) can be done on a microchip without compromising the separation efficiency The various fluid manipulation components (separation channels, valves and filters)

as well as miniaturized auxiliary instruments like power supply, detectors and pumps can be incorporated on a single microchip to allow device integration27, 28 With such integration, microchip CE devices can be developed as portable sensors that allow point-of-care or fast on-site analysis, allowing the preservation of the sample integrity

Besides being a “lab-on-a-chip”, the microchip can be custom-designed to further enhance detection sensitivity, throughput and to allow integration of detector This can be observed in the introduction of microchip with integrated potential

gradient detection (PGD), a new conductivity detector, as reported by Feng et al.29

and in μ-CAE where the microchannels are radially distributed on a small microchip

by Mathies and his coworkers25 All these can be achieved by using computer aided design softwares like AutoCAD, CorelDraw or FreeHand so as to tailor the fluid circuit on the microchip for the intended analytical methods A master template is then created so as to allow the transfer of the design directly onto the chosen microchip substrate or for further replication

However, an appropriate substrate and its complementary microfabrication technique have to be chosen before making the master template The selection of substrate is of importance as its properties, such as the charges on the microchannel’s surface, electrical conductivity, thermal insulation, optical clarity and solvent compatibility as well as the availability of established modifications/surface chemistry

of the substrate, can significantly affect the MCE’s separation capability and efficiency30 Moreover, the physical properties of the substrate like rigidity, glass transition temperature, melt temperature and thermal expansion coefficient need to be

considered in deciding the type of microfabrication technology to be used as well as microfabrication parameters, like the thickness of the photoresist layer to be applied, the duration of UV exposure and wet etching, to be optimized31

There are mainly two types of microchip substrates to be considered – rigid glass and silicon or elastomeric polymers Glass substrate is commonly known for its good optical clarity and good solvent compatibility In addition, it has a stable microchannel surface that gives rise to reproducible EOF closely resembling that of the fused silica capillary32, 33 Due to the rigid physical property, micromachining technique, which involves photolithography or electron beam lithography and etching,

is utilized to fabricate glass and Si microchips Such a technique is stringent and tedious owing to the need for a clean room facility Furthermore, glass substrate is fragile and requires delicate handling Hence, the microfabrication of such microchip

is expensive and makes it cost inefficient to be disposable

Polymer based microchips are, thus, preferred in both the research and industrial fields These polymers can be moulded readily with simplified microfabrication process and thus allowing the mass production of such microchips There are generally three classes of polymers with varying rigidity – elastomeric polymers, duroplastic polymers and thermoplastic polymers34 Elastomeric polymers like polydimethylsiloxane (PDMS) and perfluoropolyethers (PFPEs) are weakly cross-linked polymer chains that will return to its original state even after it is deformed by the application of external forces Hence, soft lithography technique is commonly utilized for the microfabrication of such microchips35 The design of the fluid circuit is first printed on a transparency or chrome mask The smallest feature size of the former is limited to only 8 μm while the latter’s, which is more costly, can

be further reduced36 Photolithography is typically used to transfer the fluid circuit

design to a silicon substrate which is used as a master template for replica moulding

of multiple microchips Such a technique allows multi-layering of the elastomers thereby creating a three-dimensional (3-D) microchip system With the simplicity of such technique, soft lithography enables one to vary the design of the fluid circuit with ease

A similar technique to soft lithography is also used to fabricate duroplastic based microchip37 Duroplastic polymers such as thermoset polyester, resist materials and polyimide are more strongly cross-linked Thus, it is harder to re-mould them A refined soft lithography technique has to be used instead Its difference from soft lithography lies in that the polymer is partially cured using UV light before removing

it from the template The final product is then obtained with complete curing against another partially cured polymer, thereby providing a good sealing between polymers

Polymethylmethacrylate (PMMA), polycarbonate (PC), cyclic olefin copolymer (COC), polystyrene (PS), polyvinylchloride (PVC) and polyethyleneterephthalate glycol (PETG) are examples of thermoplastic polymers Like elastomeric polymers, they are formed from weakly linked chain molecules The moulding of such polymers requires the careful manipulation of the polymer’s glass transition temperature (Tg) As such, embossing38 and injection moulding39 are more suitable microfabrication techniques for this class of polymeric microchips Embossing involves the use of pressure and heat with hydraulic vacuum pumps to pattern the polymers against the master silicon or metal template The silicon template can be constructed from the previously mentioned micromachining method while the metal stamps are either electroplated from the silicon masters or manufactured with the LIGA (lithography, electroplating and moulding in German) process39, 40 Although embossing procedure seems uncomplicated, the template making process is

time consuming and limiting Furthermore, only mono-layer planar microchips can be obtained and an initial costly capital investment in the equipment is needed Hence, such a technique is only suitable for routine production of proven microchip designs Alternatively, injection moulding can also be used for making these polymeric microchips It involves the use of the melted pre-polymerized pellets of the thermoplastic polymers before injecting them into a heated mould cavity under high pressure This is followed by the release of the polymer from the mould after cooling

it to below Tg It is sometimes preferred over embossing as it allows for higher throughput and is thus more efficient in mass production

Beside the various microfabrication techniques as described above, laser ablation41 can also be used to create the fluid circuit designs on the thermoplastic polymers A high-powered pulsed laser, like ArF excimer laser (193 nm), KrF (248 nm) and the CO2 lasers, incised the designs onto the substrate Such a technique allows fast fabrication of newly designed microchip since the design can be directly inputted into the microfabrication system to allow direct translation of the design onto the substrate Unfortunately, it is unsuitable for mass production because of the inherent serial nature of the system36

With the numerous benefits and the wide variety of substrates and techniques available for microfabrication of microchips, it is of no doubt that microchip can be a potentially useful tool that can aid in the advancement of various research fields like the life science, clinical analysis and biomedicine The possibility of “lab-on-a-chip”

on a single platform, fast analysis results, high throughput and the availability of biocompatible polymers like PDMS coupled with relatively low cost of production will continue to attract researchers in these fields towards MCE

1.3 Analysis of small biomolecules

Biomolecules refer to molecules that are formed naturally from various biological processes, like metabolism and biosynthesis, in living organisms They are comprised primarily of carbon, hydrogen, nitrogen, oxygen, phosphorous and sulfur

of varying molecular weight that range from small biomolecules like amino acids, catecholamine neurotransmitters, polyamines, hormones, nucleosides and nucleotides

to macrobiomolecules like proteins, deoxyribonucleic acids (DNA) and polysaccharides The analysis of larger biomolecules, that are separated based on the differences in molecular weights, is so well established that techniques like gel electrophoresis or CE with electrolytes containing sieving matrices are commonly used by most researchers when they encounter such analytes42-46 With these techniques, structural, conformational and biological information of macrobiomolecules can be obtained Conversely, the study on small biomolecules is often neglected since they are regarded to be too small to contain any useful genetic information But these small biomolecules are the building blocks needed for biosynthesis of macrobiomolecules, intermediates of metabolism or cofactors of biochemical processes Any abnormality occurring to these biomolecules is usually an indication of the occurrence of diseases As such, the analysis of these biomolecules enables the detection of early onset of diseases (i.e malfunctioning metabolism or biosynthesis system), to control and monitor their progress as well as to obtain information for drug discovery Consequently, these biomolecules are being investigated as biomarkers of potential diseases

Biomarkers are biomolecules that are subjected to cellular, biochemical, molecular or genetic alterations such that a biological process can be recognized and monitored47 When the biological process is disrupted, the level of biomarkers will be

unusual For instance, free modified nucleosides are commonly found posttranscriptional in ribonucleic acids (RNA) However, when a patient succumbs to cancer, the quantity of these modified nucleosides found in his/her body is significantly higher than a healthy patient48 These biomarkers are commonly retrieved from body fluids like serum, urine and cerebrospinal fluids These fluids are complex matrices due to the presence of other biomolecules like proteins, urea and carbohydrates that will interfere in their analysis In addition, the low levels of these biomarkers in body fluids can lead to difficulties in their detection in these fluids For

instance, some amino acids in urine were reported by Soga et al.49 to be as low as 13 μmolL-1

As such, it is necessary to develop sufficiently sensitive analytical methods that can be dedicated to high throughput routine analysis (both qualitative and quantitative) of these small biomolecules Several methods, including high-performance liquid chromatography-mass spectrometry (HPLC-MS)50, gas chromatography-mass spectrometry (GC-MS)51, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS)52 and CE47, have been developed for the analysis of small biomolecules Among these methods, CE, coupled with different detection methods that include laser induced fluorescence (LIF), UV, conductivity and MS, is thought to have many advantages over other methods 47, 49, 53-

57 The small biomolecules are typically charged which will facilitate their separation

in CE in view of the latter’s inherent ability of separating charged molecules based on size-to-charge ratio Moreover, the equipment and operational cost of CE, compared

to the abovementioned methods, is lower without sacrificing separation efficiency In addition, minimal sample preparation is required for CE analysis Thus, it will be

ideal for routine analysis, e.g in clinical analysis, where large numbers of samples need to be examined

1.4 Project objectives

The quantitative analysis of small biomolecules allows better understanding of

a patient’s health Any unusual changes in their concentrations in the body system raise the alarm of potential health problem Unfortunately, these small biomolecules are present in small amounts in the human body such that their analyses are often laborious due to the need for extensive sample preparation and sensitive detection method Such problems also impede routine analysis However, with the high separation efficiency that can be expected from CE and its miniaturization, it has the potential to overcome many problems encountered in the analyses of small biomolecules Hence, a study on the use of CE and MCE for the analyses of small

biomolecules will be carried out in this work

In Chapter 2, a target specific liquid-liquid extraction of an endogenous nucleoside, adenosine, was investigated The extraction served to aid in improving the detection of adenosine via pre-concentrating the adenosine in a small volume of extractant An ionic liquid, a tunable stable solvent with negligible vapour pressure, was utilized as an extractant in place of the toxic volatile organic solvents in this extraction The aptamer of adenosine, a polynucleotide with structural recognition for adenosine, was further added into the ionic liquid extractant to assess any improvement in the latter’s extraction for adenosine, a biomarker for inflammatory diseases and cell stress The study of the extraction efficiency of these extractants was carried out with simple CE-UV technique due to adenosine’s UV-absorbing nature

In view of the non-UV absorbing property of many small biomolecules like amino acids and biogenic amines as well as the need for rapid analysis, a novel contact conductivity detection system for microfluidic devices was developed Its working principles and analytical performances were described in Chapter 3 This detector served to provide a universal mode of detection while the microfluidic device aided in enhancing the analytical throughput Its analytical performance was evaluated initially with simple metal ions, before it was utilized in the separation of amino acids and biogenic amines

1.5 References

(1) Tiselius, A In Nova Acta Regia Sociates Scientiarum Upsaliensis, 1930, Ser

IV, Vol 7, Number 4; Vol 7

(2) Hjerten, S Chromatogr Rev 1967, 9, 122-219

(3) Jorgenson, J W.; Lukacs, K D Anal Chem 1981, 53, 1298-1302

(4) Baker, R D Capillary Electrophoresis; John Wiley & Sons, Inc, 1995

(5) Guan, Y Q.; Chu, Q C.; Fu, L.; Wu, T.; Ye, J N Food Chem 2006, 94,

157-162

(6) Hutchinson, J P.; Evenhuis, C J.; Johns, C.; Kazarian, A A.; Breadmore, M

C.; Macka, M.; Hilder, E F.; Guijt, R M.; Dicinoski, G W.; Haddad, P R

(12) Powell, P R.; Ewing, A G Anal Bioanal Chem 2005, 382, 581-591

(13) Kang, D.; Chung, D S.; Kang, S H.; Kim, Y Microchem J 2005, 80,

121-125

(14) Manz, A.; Harrison, D J.; Verpoorte, E M J.; Fettinger, J C.; Paulus, A.;

Ludi, H.; Widmer, H M J Chromatogr 1992, 593, 253-258

(15) Soper, S A.; Henry, A C.; Vaidya, B.; Galloway, M.; Wabuyele, M.;

McCarley, R L Anal.Chim Acta 2002, 470, 87-99

(16) Belder, D.; Ludwig, M Electrophoresis 2003, 24, 3595-3606

(17) Makamba, H.; Kim, J H.; Lim, K.; Park, N.; Hahn, J H Electrophoresis 2003,

(18) Lichtenberg, J.; de Rooij, N F.; Verpoorte, E Talanta 2002, 56, 233-266 (19) Greenwood, P A.; Greenway, G M TrAC, Trends Anal Chem 2002, 21,

726-740

(20) Jakeway, S C.; de Mello, A J.; Russell, E L Fresen J Anal Chem 2000,

366, 525-539

(21) McClain, M A.; Culbertson, C T.; Jacobson, S C.; Allbritton, N L.; Sims, C

E.; Ramsey, J M Anal Chem 2003, 75, 5646-5655

(22) Effenhauser, C S.; Manz, A.; Widmer, H M Anal Chem 1993, 65,

2637-2642

(23) Dolnik, V.; Liu, S R.; Jovanovich, S Electrophoresis 2000, 21, 41-54

(24) Simpson, P C.; Roach, D.; Woolley, A T.; Thorsen, T.; Johnston, R.;

Sensabaugh, G F.; Mathies, R A Proc Natl Acad Sci USA 1998, 95,

2256-2261

(25) Emrich, C A.; Tian, H J.; Medintz, I L.; Mathies, R A Anal Chem 2002,

74, 5076-5083

(26) Jin, L J.; Ferrance, J.; Landers, J P Biotechniques 2001, 31, 1332-1353

(27) Renzi, R F.; Stamps, J.; Horn, B A.; Ferko, S.; VanderNoot, V A.; West, J

A A.; Crocker, R.; Wiedenman, B.; Yee, D.; Fruetel, J A Anal Chem 2005,

77, 435-441

(28) Roman, G T.; Kennedy, R T J Chromatogr A 2007, 1168, 170-188

(29) Feng, H T.; Wei, H P.; Li, S F Y Electrophoresis 2004, 25, 909-913

(30) Shadpour, H.; Musyimi, H.; Chen, J F.; Soper, S A J Chromatogr A 2006,

1111, 238-251

(31) Becker, H.; Locascio, L E Talanta 2002, 56, 267-287

(32) Manz, A.; Fettinger, J C.; Verpoorte, E.; Ludi, H.; Widmer, H M.; Harrison,

D J TrAC, Trends Anal Chem 1991, 10, 144-149

(33) Harrison, D J.; Manz, A.; Fan, Z H.; Ludi, H.; Widmer, H M Anal Chem

1992, 64, 1926-1932

(34) Becker, H.; Gartner, C Electrophoresis 2000, 21, 12-26

(35) Whitesides, G M.; Ostuni, E.; Takayama, S.; Jiang, X Y.; Ingber, D E An

Rev Biomed Eng 2001, 3, 335-373

(36) Fiorini, G S.; Chiu, D T Biotechniques 2005, 38, 429-446

(37) Fiorini, G S.; Lorenz, R M.; Kuo, J S.; Chiu, D T Anal Chem 2004, 76,

4697-4704

(38) Kricka, L J.; Fortina, P.; Panaro, N J.; Wilding, P.; Alonso-Amigo, G.;

Becker, H Lab Chip 2002, 2, 1-4

(39) McCormick, R M.; Nelson, R J.; AlonsoAmigo, M G.; Benvegnu, J.;

Hooper, H H Anal Chem 1997, 69, 2626-2630

(40) Galloway, M.; Stryjewski, W.; Henry, A.; Ford, S M.; Llopis, S.; McCarley,

R L.; Soper, S A Anal Chem 2002, 74, 2407-2415

(41) Klank, H.; Kutter, J P.; Geschke, O Lab Chip 2002, 2, 242-246

(42) Rickwood, D.; Hames, B D Gel Electrophoresis of Nucleic Acids - A

Practical Approach; IRL Press, Oxford: Washington, DC, 1982

(43) Andrews, A T In Electrophoresis - Theory, Techniques, and Biochemical and

Clinical Applications; Clarendon Press, Oxford, 1981

(44) Dolnik, V.; Liu, J P.; Banks, J F.; Novotny, M V.; Bocek, P J Chromatogr

1989, 480, 321-330

(45) Righetti, P G.; Gelfi, C J Biochem Biophys Methods 1999, 41, 75-90

(46) Xu, F.; Baba, Y Electrophoresis 2004, 25, 2332-2345

(48) Liebich, H M.; Muller-Hagedorn, S.; Klaus, F.; Meziane, K.; Kim, K R.;

Frickenschmidt, A.; Kammerer, B J Chromatogr A 2005, 1071, 271-275

(49) Soga, T.; Kakazu, Y.; Robert, M.; Tomita, M.; Nishioka, T Electrophoresis

2004, 25, 1964-1972

(50) Tuytten, R.; Lemiere, F.; Van Dongen, W.; Witters, E.; Esmans, E L.;

Newton, R P.; Dudley, E Anal Chem 2008, 80, 1263-1271

(51) Shama, N.; Bai, S W.; Chung, B C.; Jung, B H Rapid Communications in

Mass Spectrometry 2008, 22, 959-964

(52) Vaidyanathan, S.; Goodacre, R Rapid Communications in Mass Spectrometry

2007, 21, 2072-2078

(53) Shihabi, Z K.; Friedberg, M A Electrophoresis 1997, 18, 1724-1732

(54) Kovacs, A.; Simon-Sarkadi, L.; Ganzler, K J Chromatogr A 1999, 836,

CHAPTER 2 Capillary Electrophoresis Analysis of Adenosine

2.1 Importance of the analysis of adenosine

Adenosine is an endogenous nucleoside that can be found in micromolar concentration in biological fluids like urine, synovial fluid and cerebrospinal fluid It

is mainly produced from the 5’-nucleotidase catalyzed dephosphorylation of the adenosine triphosphate (ATP) within the cells1 Adenosine is subsequently released from the cells and interacts with cell receptors, such as adenosine P1, in the blood circulation system In these interactions, adenosine behaves as a signaling molecule

by exerting several physiological effects on various cells2, 3 Besides being a signaling molecule, adenosine has immunosuppressive properties towards white blood cells and

immune system related cells Sottofattori et al.2 reported that adenosine was responsible for the development of inflammatory diseases, for example, rheumatoid arthritis Further to its involvement in these biological functions, adenosine had been

pointed out by Abou El-Nour et al.4 to be a potential marker for cell stress since its level heightened during periods of oxygen deficiency In consideration of the biological importance of adenosine, it is thus necessary to explore various analytical methods that allow accurate quantification of adenosine present in the body fluids

Several publications have reported using immunoassay5, 6, radioactive labeled substrates coupled to scintillation counter7, high performance liquid chromatography (HPLC)2, 3 and liquid chromatography-mass spectrometry (LC-MS)8for the determination of adenosine in body fluids However, some of these analytical methods are health hazardous, expensive, time consuming and require extensive sample pre-treatment Consequently, CE has emerged to be a potentially fast and sensitive alternative technique for the detection of adenosine1, 9, 10 In addition, its

isotope-instrumental set-up is simple and relatively inexpensive Low reagent consumption in

CE analysis reduces the need for large volumes of hazardous volatile organic compounds (VOCs) that are often required in LC instruments Moreover, its short analysis time may prove to be advantageous for routine analysis of large amounts of clinical samples However, prior to analysis, simple sample pre-treatment has to be carried out to further enhance the sensitivity of the technique and to isolate the analyte from the complex biological matrix

2.2 Liquid-liquid extraction of adenosine

2.2.1 Liquid-liquid extraction using ionic liquid

Besides adenosine, biological fluids also contain many other biomolecules, ranging from small molecules (for example metabolites) to large molecules (for example proteins) which can interfere with the quantitative determination of adenosine during CE analysis Sample pre-treatment is, thus, needed to enhance the detection sensitivity of adenosine Most importantly, pre-treatment ensures more reproducible determinations of adenosine A poor sample pre-treatment can invalidate the entire analytical method, leading to erroneous results and waste of time11

There are a wide variety of sample preparation techniques that have been developed to date11-13 One such method is the liquid-liquid extraction (LLE) technique The LLE is commonly used in the industries in view of its simplicity, low costs and ease of scaling up Studies on using LLE to pre-concentrate and purify biomolecules have also been carried out extensively14-16 In LLE, the analyte of interest is transferred from the sample matrix to an immiscible solvent, known as extractant, in which the analyte has preferential solubility in17, 18 Despite its

widespread use, critics of LLE have spoken against it mainly for the use of large volumes of VOCs as solvents These VOCs are hazardous to both the environment and health due to their toxicities as well as flammabilities19 In addition, these VOCs are unsuitable for the purification of biomolecules since it may cause them to denature17

Hence, an alternative to these VOCs should be used instead while ensuring the benefits of LLE can still be realized In 1998, Rogers and his co-workers20 initiated the use of ionic liquids (ILs) in the LLE of substituted benzene derivatives from water They reported the inherent characteristics of ILs, such as negligible vapour pressure, good air and water stability, wide liquid range as well as the ability to tune their properties to solvate a wide range of compounds which would make them desirable

substitutes in LLE Moreover, it had also been reported, by both Hiroyuki et al.21 and

Winterton et al.22, that ILs could preserve biomolecules instead of denaturing them Thus, this had subsequently led to a series of publications in the analysis of environmental pollutants23-25, food contaminants26 and small biomolecules like amino acids27 using ILs as an extractant in LLE

The ionic liquids (ILs), otherwise known as molten salts, belong to a class of non-molecular ionic solvents with melting points of not exceeding 100 ˚C 28 They consist typically of a bulky asymmetric organic cation such as those depicted in Figure 2.1, coupled with an assortment of anions, for instance, [PF6]-, [BF4]-, [CF3SO3]-, [(CF3SO2)2N]-, [CF3CO2]-, [CH3CO2]-, [NO3]-, [Cl]- and [Br]- 19, 29 The synthesis of ILs consists of two main parts – protonation or alkylation to form the cationic moiety and anionic exchange30 By varying the length of the alkyl substituents on the cations as well as the nature of the complementary anion, various properties of ILs such as viscosity, hydrophilicity and solvation power can be tailored

to fit the analyst requirements However, the conventional ILs synthesis process usually takes 2-3 days due to a relatively longer time needed for the formation of cation Fortunately, this synthesis time can be reduced to mere minutes, with minimal

starting reagents and satisfactory product yield, via microwave-assisted synthesis as reported by Deetlefs et al.31

N

N

1-Butyl-3-methylimidazolium

N N-methyl-N-butylpyrrolidinium

4-Butyl-4-methylmorpholinium

Figure 2.1 Representative cations used in the synthesis of ILs

With the ease of synthesis and the attractive characteristics of ILs, it is with little doubt that ILs are potentially suitable extractants for the LLE of adenosine However, ILs do not possess any extraction specificity Any biomolecules that have similar physical and chemical characteristics to adenosine, for instance other nucleotides such as guanosine, cytosine as well as their metabolites, in biological fluids may also be extracted by the ILs along with adenosine Additional molecular recognition properties have to be induced on the ILs so as to enhance their extraction selectivity This can be achieved by introducing aptamer into the ionic liquid

2.2.2 Target specific liquid-liquid extraction with ionic liquid-aptamer

Aptamers are short functional oligonucleotides that are used as ligands to bind

to a given target with high specificity and affinity Their dissociation constant for small targets such as Zn2+, arginine and carcinogenic aromatic amines are in millimolar to micromolar range while that of large targets, for instance proteins, reach

as low as nanomolar to picomolar range32 There are DNA and RNA aptamers The DNA aptamers are relatively more stable as they are more nuclease resistant while the RNA aptamers possess comparatively higher binding affinity due to their ability to have greater conformational flexibility32 They are selected from very large libraries

of randomized oligonucleotide sequences via in vitro selection and amplification with

techniques such as systematic evolution of ligands by exponential enrichment (SELEX) and equilibrium capillary electrophoresis equilibrium mixture (ECEEM)33 The expanding aptamer database34 for a diversity of targets have consequently been extensively used by researchers for various analytical and diagnostics applications in the fields of biological and electrochemical sensors, affinity chromatography and affinity CE32, 33, 35, 36

In these applications, the advantages of aptamers have led to promising results over antibodies37 The aptamers are chemically synthesized with high reproducibility

as well as accuracy and, thus, do not rely on animals for production Moreover, the desired aptamers can be selected and modified with fluorescence label under customized non-physiological conditions Most importantly, unlike antibodies, aptamers can undergo reversible denaturation and have longer shelf life even at ambient temperature Their good stability and the ability to customize them to suit the intended purpose meant that further applications of aptamers can be explored

Previously, there are some publications reporting on work involving deoxyribonucleic acids (DNA) and ILs 21, 38-40 As such, it is possible to conjugate aptamers to ILs to induce molecular recognition specificity on the latter Hence, in this work, a preliminary investigation was carried out to evaluate the extraction efficiency of a target specific ILs extractant to isolate adenosine from aqueous sample Two methods of conjugating the aptamer to ILs were proposed

The first method involved conjugating 1-butyl-3-methylimidazolium ([C4MIM]) based ILs to the negatively charged oligonucleotides based aptamer of adenosine via acid-base neutralization Ohno and his coworkers39 had earlier reported

on using basic 1-butyl-3-methylimidazolium hydroxide ([C4MIM]OH)), as shown in Figure 2.2(a), to neutralize the protonated phosphate groups on the double stranded DNA (dsDNA) as depicted in Figure 2.2(b) Hence, a slightly modified procedure where an aptamer, a single stranded DNA (ssDNA), was used in place of the dsDNA, was adopted in this work

The second method involved the dissolution of aptamer in methylimidazole based ILs Several papers had reported on the solubility of synthetic polymers like poly(N,N-dimethylacrylamide) and poly(acrylonitrile) in butyltrimethylammonium bis(triflylmethylsulfonyl)imide and 1-alkyl-3-methylimidazolium based ILs 22, 41-44 Consequently, Ohno’s and his co-workers had published works on the dissolution of natural polymers like DNA and ribonucleic acid (RNA), in ammonium, imidazolium and pyridinium cationic based ILs21, 38-40 The high ionic salt composition of ILs disrupts the higher order structure of deoxyribonuclease (DNase) and ribonuclease (RNase), causing them to be degraded and thus deactivated As such, the structures of DNA and RNA can be preserved The DNA and RNA can, hence, be handled at room temperature with ease Furthermore,

1-butyl-3-the group had also reported that 1-butyl-3-the ILs should preferably be made up of imidazolium cations coupled to halide or carboxylate anions, and hence such ILs have good solvation power for these oligonucleotides

N N N N

NH2

O

H OH

H H H H

O P

NH2N

O

H

H H H H

OH

O P HO O

H OH

H H H H

O P HO

OH O

2'-deoxycytosine-5'-monophosphate (deoxyCMP)

NH O

O N O

H OH

H H H H

O P HO OH O

-1-butyl-3-methylimidazolium hydroxide ([C4MIM]OH)

and (b) the four nucleotides on the dsDNA

In this work, a 42-nucleotide long aptamer for adenosine was first dissolved in [C4MIM] cationic based IL before it was used as an extractant in LLE to extract adenosine from an aqueous sample

spectra were recorded via a Bruker ACF 300 FT NMR spectrometer (Bruker,

Rheinstetten, Germany), with chemical shifts referenced to residual solvent peaks in the respective deuterated solvents

CE was performed on a laboratory built system equipped with a power supply (CE Resources, Singapore) and a Linear Instrument UV/Vis 200 (Reno, NV, USA) as its complementary UV/Vis detector Data acquisition and recording of electropherograms were accomplished with CSW Chromatography Station (DataApex, Prague, Czech Republic) Bare silica capillary with external polyimide coating of i.d

50 μm and o.d 360 μm (Polymicro Technologies, Phoenix, AZ., USA) were used The temperature was maintained at 25 ± 1 ˚C The pH of the buffer solutions were measured with a pH meter (Science and Medical Pte Ltd, Singapore)

2.3.1.2 Reagents and chemicals

All chemicals were of reagent grade unless othewise stated Methylimidazole (MIM) and 1-chlorobutane (BuCl) were purchased from Fluka (Buchs, Switzerland) Cytosine, hexafluorophosphoric acid (HPF6), 2’-deoxycytidine-5’-monophosphate (deoxyCMP), 2’-deoxyguanosine, thymidine and adenosine were procured from Sigma (Steinheim, Germany) Poly(vinylalcohol) (PVA) (MW ~ 50 000, 99+% hydrolyzed) was purchased from Aldrich (St Louis, USA) Duolite A113 chloride (Cl-) based resin (BDH Chemicals, Poole, UK) was obtained from the Analytical

Teaching Laboratory, Department of Chemistry, NUS while Amberlite® IRA400 hydroxide (OH-) form resin was obtained from Supelco (Pennsylvania, USA) Triethylamine (NEt3) and silver nitrate (AgNO3) were purchased from Riedel de Hặn (Seezle, Germany) Both the trishydroxymethylaminomethane (Tris) and ethylenediaminetetraacetic acid (EDTA) buffers as well as HPLC-purified 42-oligonucleotide long DNA aptamer (42-mer) (5’- GTG CTT GGG GGA GTA TTG CGG AGG AAA GCG GCC CTG CTG AAG-3’) for adenosine were procured from

1st Base (Singapore) HPLC grade ethyl acetate (EA), isopropanol (IPA), acetic acid and sodium acetate were obtained from Merck (Darmstadt, Germany) Acetonitrile (ACN) was obtained from Tedia (Fairfield, Ohio, USA) in HPLC grade Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were bought from Chemicon (Temecula, California, USA) and from Fluka BioChemika (Buch, Switzerland) respectively Deionised water (DI H2O) used throughout this experiment had resistivity ≥18 MΩ and was supplied by a NANOpure ultrapure water purification system (Barnstead, IA, USA) All solutions used in CE were filtered with 0.20 μm Millisart filter (Gottingen, Germany) prior to use

2.3.2 Microwave synthesis of 1-butyl-3-methylimidazolium chloride

1-Butyl-3-methylimidazolium chloride ([C4MIM]Cl) was synthesized via a

modified procedure published by Deetlefs et.al.31 The microwave reaction parameters were further refined in consideration that a different microwave reactor was used The modified procedure was described as followed: The distilled 1-methylimidazole and 1-chlorobutane were mixed in a molar ratio of 1:1.2 before being allowed to react at

300 ˚C for 20 min powered at 300 W under closed vessel condition A resulting golden yellow reaction mixture was obtained and transferred to a pear-shape flask It

was washed with ethyl acetate thrice under a 50 ˚C warm water bath prior to solvent

removal in vacuo The product was subsequently characterized by 1H NMR spectrometery and its purity was verified using CE-UV as mentioned in Section 2.3.3 Its 1H NMR spectrum (300 MHz, D2O, ppm), as seen in Appendix 1, was as followed:

δ = 0.8536 (t), 1.2491 (sextet), 1.7800 (quintet), 3.8180 (single), 4.1242 (triplet), 7.3544 (singlet) and 7.4031 (singlet)

2.3.3 Analysis of 1-butyl-3-methylimidazole based ionic liquid via CE-UV

A PVA coated capillary was first prepared according to the procedures

illustrated by Gilges et al.45 The running buffer, consisted of 5.0 mM sodium acetate, 5.0 mM triethylamine and 75 mM sodium chloride adjusted to pH 4.5 by acetic acid,

was similar to that reported by Qin et al.46 The capillary was rinsed with water and running buffer for 10 min each before the commencement of daily runs Samples were loaded by gravity at the anode end for 10 s The separation was carried out at a constant voltage of +14 kV at a detection wavelength of 210 nm The capillary was flushed with running buffer for 2 min in between runs The capillary was rinsed with water for 2 min and dried with air prior to storage at the end of the day

2.3.4 Synthesis of 1-butyl-3-methylimidazolium based ionic liquid

The [C4MIM]OH was obtained from [C4MIM]Cl utilizing ionic exchange chromatographic method, with anionic resins, briefly illustrated by Ohno and his coworkers39 Two types of resins, Duolite A113 (OH-) and Amberlite IRA-400 (OH-), were tried in this work

Prior to use, the Duolite A113 (Cl-) resins, were converted to Duolite A113 (OH-) resins with 0.5 M NaOH A saturated [C4MeIM]Cl solution was subsequently